We are analyzing the interactions between virus glycoproteins and their receptors for several enveloped viruses of significance for public health in the US and worldwide. Our goals are to define mechanisms of virus entry and cell tropism, identify cellular receptors that mediate virus entry, and develop novel antiviral approaches for treatment (antiviral drugs) and prevention (vaccines, microbicides). 1) Human hepesviruses. During the past year we extended our studies of CD46 as the receptor for human herpesvirus 6 (HHV-6) by demonstrating its presence and receptor functionality on glial cells. We also extended our work on Kapsosi's Sarcoma-Associated Virus (KSHV, aka human herpesvirus 8). KSHV is etiologically linked to Kaposis sarcoma, a major opportunistic disease afflicting people with AIDS. The mechanism by which KSHV enters cells is poorly understood; the limited data to date suggests that it enters via direct fusion between the envelope membrane of the virus and the plasma membrane of the target cell. We previously devised a cell fusion assay to measure the functionality of KSHV glycoproteins, and adapted this assay to identify a KSHV fusion receptor by functional selection off a cDNA library from a permissive target cell. The receptor is a previously described transport protein with 12 transmembrane segments. During the past year, we proved its role as a KSHV fusion entry by several criteria: a) Gain of function. Expression of the recombinant protein rendered otherwise negative target cells permissive for both KSHV cell fusion and virion entry. b) Loss of function. Anti-peptide antibodies against several predicted extracellular regions of the protein blocked both KSHV fusion and virion entry in target cells that were naturally permissive. c) Correlation in a panel of diverse cell types between permissiveness as fusion targets and expression of the transporter protein. The molecule unambiguously identified as a KSHV fusion/entry receptor in this work is distinct from any candidate KSHV receptors proposed thusfar. 2) Hepatitis C virus (HCV). We are particularly interested in developing systems to study HCV entry, for use in defining essential receptors and elucidating entry mechanisms, assaying HCV neutralizing antibodies in infected people and in vaccine studies. To this end, we are devising systems to produce HCV virus-like particles containing the functional HCV E1 and E2 glycoproteins and containing an RNA that encodes a reporter gene; this will enable rapid, quantitative, and sensitive measurement of entry. The first approach employs a recombinant baculovirus system, which to date is the only approach that has yielded large amounts of purified particles composed of the HCV structural genes (core, E1, E2). We have devised several approaches to encapsidate the desired reporter gene-encoding RNA. In the second approach, we are seeking to develop chimeric VLPs containing the core of West Nile virus, an ecapsidated reporter RNA, and HCV glycoproteins on the surface. We have obtained preliminary evidence that under certain conditions, HCV E1 and E2 can be incorporated into such chimeric particles, and can facilitate entry by virtue of E2 binding to CD81 on the target cell surface.